The evolution of new strains of multi drug resistant bacteria is an emerging problem. Currently, there is a lack of a concerted effort among major pharmaceutical companies for addressing this problem. Highlighting this aspect is the small number of U.S. Food and Drug Administration (FDA) approved antibiotics. For example, only Teflaro® (ceftaroline fosamil) has been approved by the FDA since 2010. Moreover, anti-biotic resistant gram-negative bacteria in hospital acquired infections are increasingly becoming problematic with dwindling supplies of antibiotics that are efficacious against these pathogens. These antibiotic resistant bacterial infections in immunocompromised/sick patients further complicate the therapy of other underlying diseases. Thus, new strategies for the development of anti-bacterial drugs and methods of treatment are needed.
Accordingly, a paradigm shift in how the scientific community thinks about developing new anti-bacterial therapeutics may be underway. Fox, Nature Biotechnology 31(5): 379-382 (2013). This shift is into the potential use and therapeutic potential of anti-microbial peptides (AMPs). AMPs are molecules naturally found in microorganisms to mammals that comprise short peptides of about 15 amino acids to about 50 amino acids. The product is ribosomally synthesized and often post-translationally modified. This synthesis is in contrast to other natural peptide based antibiotics, such as vancomycin, which are formed by enzymatic cleavage. Fox, Nature Biotechnology 31(5): 379-382 (2013).
Anti-microbial peptides serve as a major component of the innate immunity and complement cell-mediated immune responses. The mechanism of action of most anti-microbial peptides depends on the target cellular surface. The principle anti-microbial activity of most AMPs has been proposed by the so called “carpet model,” wherein the amino acid composition, amphipathicity, and positive charge allow the peptides to electrostatically interact and insert into the mostly negatively charged microbial cell membranes, resulting in pore formation and cell death. Other modes of action include the inhibition of cell wall synthesis, autolysin activation, and the inhibition of DNA, RNA, and protein synthesis. The specificity of AMPs to microbes is due to mammalian and plant cell membranes having no net charge, which limits AMP interaction. Furthermore, acquired resistance to AMPs appears to be low as only a moderate increase in AMP resistance was observed after more than ten passages. Hancock, The Lancet Infectious Diseases 1(3): 156-164 (2001).
Additional problems emerging in the treatment of infectious diseases is biofilms. Sanchez et al., BMC Infectious Diseases 13: 47 (2013); Dean et al., BMC Microbiology 11: 114 (2011). Bacteria and fungi can assume a planktonic or free swimming phenotype, or they can exist as part of a colony (i.e., attached to a surface) or a biofilm. A colony of bacteria in a biofilm are surrounded by an exopolysaccharide matrix for protection, making them notoriously difficult to eradicate. Biofilms are found in 60-80% of chronic hospital-associated infections. Van Acker et al., Trends in Microbiology 22(6): 326-333 (2014). There are endogenous anti-biofilm peptides, Dean et al., BMC Microbiology 11: 114 (2011); de la Fuente-Nunez et al., PLoS Pathogens 10(5): e1004152 (2014), and while these peptides are similar to AMPs, not all of them are anti-microbial. These anti-biofilm peptides are potentially useful as topical agents for bacterial infections with a biofilm component, but in their natural form, they are not amenable for the treatment of systemic infections. Dean et al., BMC Microbiology 11: 114 (2011).
AMPs have principally been generated or discovered from naturally occurring AMPs. For example, U.S. Pat. No. 7,550,558 describes a method of using phage display to identify new anti-microbial peptides. Using the methods of this invention, the identified and most efficacious peptide had a high level of similarity to naturally occurring indolicidin and tritrpticin AMPs, which are known to have high levels of in vivo toxicity. While this AMP is potentially useful, no in vivo safety testing or efficacy was provided. U.S. Pat. No. 7,452,864 describes the use of a natural peptide cathelicidin LL-37 for topical wound regeneration promoted by its anti-microbial activity. Further examples of naturally derived peptides that have promising in vitro results are described by U.S. Pat. Nos. 7,531,509 and 7,504,380. However, anti-microbial peptides based on native peptide sequences are often subject to in vivo proteolysis or degradation, preventing in vivo efficacy. Fox, Nature Biotechnology 31(5): 379-382 (2013).
Thus, despite the promise of AMPs, many concerns remain, including toxicity, oral availability, and in vivo stability. Many peptides lack activity against gram-negative bacterial pathogens. Fox, Nature Biotechnology 31(5): 379-382 (2013). AMPs can have toxic effects from their hemolytic activity. Furthermore, naturally occurring small AMP-like melanocortin peptides have been shown to have potent cardiovascular side-effects from the presence of “RFamide” sequences (Arg-Phe or conservative substitutions) near the C-terminus of these peptides. Thiemermann et al., Biochem Biophys Res Commun. 175(1): 318-324 (1991); Gruber et al., Am J Physiol. 257(4 Pt 2): R681-694 (1989). U.S. Pat. No. 8,541,545 describes the elimination of RFamide effects in a similar class of small melanocortin peptides leading to decreased cardiovascular side effects. However, no concerted effort has been made in the prior art to eliminate potential RFamide effects in AMPs.
Thus, described herein, are rationally designed AMPs with efficacy against both gram negative and gram positive bacteria. The AMPs described herein further have modifications to overcome previous limitations of AMPs, which include decreased proteolysis, reduced hemolysis, and increased in vivo safety. Further, described herein are AMPs having both anti-biofilm and anti-microbial activity in a single molecule. Additionally, contemplated herein, are methods of use of the described AMPs.